CN117126957A

CN117126957A - Detection method of China rose botrytis

Info

Publication number: CN117126957A
Application number: CN202311061853.1A
Authority: CN
Inventors: 吴红芝; 袁文斌; 金寿林; 高俊平; 李思思; 贺水莲; 马男; 孙小明; 张钊
Original assignee: China Agricultural University; Yunnan Agricultural University
Current assignee: China Agricultural University; Yunnan Agricultural University
Priority date: 2023-08-22
Filing date: 2023-08-22
Publication date: 2023-11-28

Abstract

The application relates to a detection method of Chinese rose gray mold bacteria, in particular to a detection method of Chinese rose gray mold bacteria of RPB2 genes obtained based on whole genome sequencing. The detection method comprises the following steps: obtaining the nucleotide sequence of the whole genome DNA of the sample to be detected by using a whole genome sequencing technology; performing RPB2 gene comparison on the sample to be tested; and judging the sample to be tested according to the comparison result. The application utilizes whole genome sequencing to obtain the RPB2 gene to identify the B.cinerea, thereby greatly improving the accuracy of the RPB2 gene sequence and improving the classification identification efficiency. The application adopts whole genome sequencing to obtain the RPB2 gene sequence for the first time, and discovers that the B.cinerea can be identified rapidly and accurately by utilizing the RPB2 gene.

Description

Detection method of China rose botrytis

Technical Field

The application relates to the technical fields of gardening crop disease detection and molecular biology, relates to a detection method, and in particular relates to a detection method of China rose botrytis.

Background

The sexual stage of botrytis Botrytis cinerea Pers is Botryotinia fuckeliana de Bary, which belongs to botrytis fungi of the phylum ascomycotina, and gray mold caused by botrytis infection is an important worldwide plant fungal disease. Botrytis is considered the second largest phytopathogenic fungus worldwide next to Pyricularia oryzae, and has attracted extensive attention from students worldwide. It was originally thought that Botrytis cinerea (b. Cinerea) could infect more than 200 plants, but now a total of over 1400 are known. Cinerea can infect various tissues and organs of plants, causing necrosis of organs or death of plants, resulting in serious losses of many important commercial crops, especially plantation and greenhouse crops, both pre-harvest and post-harvest. Cinerea has a variety of infection patterns and a wide host range, and the hosts of b.cinerea include fruits (such as grapes, kiwi fruits, strawberries and apples), vegetables (such as tomatoes, cucumbers, peppers and lettuce), ornamental flowers (such as China rose, lily, tulip and carnation), and the like. Cinerea can also burst at low temperature, and serious gray mold diseases of China rose can be caused, so that significant losses of cut flowers and China rose before and after picking are caused. Therefore, the accurate identification of the pathogenic bacteria of the gray mold of China rose is particularly important.

Morphology and culture characterization table of cinereaExhibiting high diversity, paul (1929) has determined by observation that B.cinerea has three morphological types: sclerotium form (Sc), spore-forming form (SD) and hypha form (M), these characteristics were subsequently applied in large numbers for the identification of b.cinerea. However, morphological features among certain species of botrytis have a high degree of similarity and these features are extremely susceptible to internal nutrition and external environmental conditions, so it is difficult to distinguish some species of botrytis using morphological features. For example, B.cinerea is very similar in its culture characteristics to B.fabae and B.calthae. However, these difficulties can be overcome by molecular methods, one of which is by analysis of one or more short universal DNA sequences to identify species. The method has the advantages of rapidness and accuracy, and is widely used for identifying and classifying species. Researchers at home and abroad have successfully developed a variety of specific DNA fragments and detection methods for identification of a variety of pathogenic fungi including botrytis, of which the most commonly used are ribosomal RNA gene Large Subunit (LSU) and Internal Transcribed Spacer (ITS) sequence fragments and three single copy nuclear gene (nDNA) fragments encoding glyceraldehyde-3-phosphate dehydrogenase (G3 PDH), heat shock protein 60 (HSP 60) and DNA-dependent RNA polymerase subunit II (RPB 2), respectively. In the past, the method of constructing phylogenetic tree by combining or separately adopting three gene sequences of RPB2, G3PDH and HSP60 can identify most species of Botrytis. However, in the past, the combination of three gene sequences of RPB2, G3PDH and HSP60 could not be used to accurately identify the 4 species of B.cinerea, B.eucalypti, B.pelargonii and B.fabae (Brauna-Etc., 2023; garfinkel,2021; kevin et al, 2014). In addition, according to the study of Staats et al (2005), accurate identification of b.cinerea, b.pelargonii and b.fabae cannot be performed with RPB2 sequences alone; neither G3PDH nor HSP60 sequences alone can accurately identify B.cinerea and B.pelargonii.

The aforementioned methods for the intervarietal identification of botrytis are not ideal in accuracy and cannot effectively identify a few species including b.cinerea. The past methods of identification by sequence alone or in combination have utilized indirect detection methods of sequencing after PCR amplification, and are not accurate enough for use in intervarietal identification of Botrytis.

Furthermore, there are differences in one aspect due to understanding to those skilled in the art; on the other hand, since the applicant has studied a lot of documents and patents while making the present application, the text is not limited to details and contents of all but it is by no means the present application does not have these prior art features, but the present application has all the prior art features, and the applicant remains in the background art to which the right of the related prior art is added.

Disclosure of Invention

Cinerea can cause serious gray mold damage of China rose, so that great loss is caused before and after the cut flower China rose is picked, and therefore, the accurate identification of the pathogenic bacteria of the gray mold of China rose is particularly important. The traditional biochemical method needs to carry out a series of complicated biochemical tests, takes a long time, and results obtained under partial conditions are difficult to accurately judge. Furthermore, the morphology and culture characteristics of cinerea show a high diversity, and it is difficult to distinguish some species of botrytis in the identification test. Current molecular biology methods are relatively simple to operate, for example, by amplifying DNA sequences of rDNA ITS segments, reference strain base sequence similarity, and performing phylogenetic relationship analysis to identify strains. However, the indirect detection method using sequencing after amplification has drawbacks in that: in the PCR amplification process of tens of cycles, it is difficult to ensure that any point of the molecular fragment is not mismatched, i.e., the result obtained by sequencing the amplified PCR fragment may deviate from the original result; on the other hand, although the current sequencing accuracy reaches 98.5% or more within a certain range, certain errors exist. That is, sequencing after PCR amplification may result in changes to the original sequence at least at two levels. The fundamental basis for the classification and identification by using the molecular diagnosis method is the difference of nucleotide base arrangement sequences, and the final sequencing result is ensured to be an important standard of the molecular diagnosis method. Although the method of sequencing after PCR amplification is widely used in classification or identification of pathogenic bacteria, the method has a possibility that nucleotide base sequences deviate from the original results in performing PCR amplification as well as sequencing. To a certain extent, the method of sequencing after PCR amplification has a certain effect on classification and identification, but the resolution at various levels within the same genus is insufficient, for example, effective identification of a small number of species including b.cinerea cannot be performed, and even if the result can be obtained, the identification result is very ambiguous, and the judgment of the test result is seriously affected.

In addition, in the past, the conventional method is to infer the species to which the query sequence belongs from the sequence with the highest similarity among the returned sequences, and the application of the method takes a long time and has insufficient discrimination for some closely related species sequences. Thus, the prior art has significant shortcomings in terms of detection methods or classification and identification methods of pathogenic bacteria. Especially when the economic crops are infected with diseases, if pathogenic bacteria cannot be accurately identified, serious losses are caused.

Aiming at the defects of the prior art, the application provides a detection method of Botrytis cinerea, which comprises the following steps: obtaining the nucleotide sequence of the whole genome DNA of the sample to be detected by using a whole genome sequencing technology; performing RPB2 gene comparison on a sample to be tested; and judging whether the sample to be tested contains the Chinese rose gray mold bacteria or not according to the comparison result. Preferably, the Botrytis cinerea is one or more of Botrytis cinerea, botrytis petingoni and Botrytis fabae. Preferably, the Botrytis cinerea is Botrytis cinerea, botrytis petingonii. Preferably, the Botrytis cinerea is Botrytis cinerea, botrytis Fabae. Preferably, the Botrytis cinerea is Botrytis cinerea. Preferably, the Botrytis cinerea is Botrytis cinerea, botrytis petingonii, botrytis fabae.

The phylogenetic tree constructed based on the RPB2 gene obtained by whole genome sequencing shows that all 100 separated strains separated and detected from China rose are B.cinerea, and the B.cinerea, B.fabae, B.eucalypti and B.pelargonii of Botrytis can be clearly distinguished in the phylogenetic tree constructed based on the RPB2 gene obtained by whole genome sequencing, but the LSU sequence fragment obtained by whole genome sequencing or the ITS sequence fragment obtained by whole genome sequencing can not achieve the purpose; on the other hand, conventional joint analysis using LSU, ITS and RPB2 sequences also fails to distinguish between several species B.cinerea, B.fabae, B.eucalypti and B.pelargoni. The RPB2 gene obtained by whole genome sequencing can accurately distinguish B.cinerea from other species of Botrytis, and a clear identification result is obtained. The detection method takes shorter time and the classification and identification results are more accurate.

Preferably, the method of comparison is: and comparing the nucleotide sequence of the whole genome DNA of the sample to be detected with the standard sequence of the RPB2 gene of the Botrytis cinerea. Preferably, the standard sequence of the RPB2 gene is shown in SEQ ID No.4.

Preferably, when the sample to be tested has an RPB2 gene and the RPB2 gene contains a specific SNP locus, the sample to be tested is indicated to belong to the Botrytis cinerea, wherein the nucleotide sequence of the RPB2 gene of the sample to be tested is shown as SEQ ID No.1 to SEQ ID No.3.

Preferably, the SNP site comprises:

SNP1 is base A or G at 282 th site from the 5' end of the nucleotide sequence shown in SEQ ID No.1 to SEQ ID No. 3;

SNP2, which is a 354 th base A or G from the 5' end of the nucleotide sequence shown in SEQ ID No.1 to SEQ ID No. 3;

SNP3 is base C or T at 468 th position from 5' end of the nucleotide sequence shown in SEQ ID No. 3;

SNP4 is base T or G at 855 th position from the 5' end of the nucleotide sequences shown in SEQ ID No.1 and SEQ ID No. 3;

SNP5, which is the 981 th base C or T from the 5' end of the nucleotide sequence shown in SEQ ID No.1 to SEQ ID No. 3;

SNP6 is base C or A at 1086 th from the 5' -end of the nucleotide sequence shown in SEQ ID No.1 to SEQ ID No.3.

Botrytis cinerea cannot be identified in the prior art, and the Botrytis cinerea can be accurately and efficiently identified through the RPB2 gene. The application discovers that the RPB2 gene may have inter-species difference through repeated experiments, which may also be the reason why the RPB2 can independently identify Botrytis cinerea.

Preferably, the step of obtaining the sample to be measured is as follows: collecting cut flower China rose tissues infected with gray mold diseases; culturing pathogenic bacteria, separating and purifying; obtaining a strain to be detected after preliminary identification; and (5) preserving.

Preferably, the test sample is incubated at 25℃and 80% relative humidity in the dark for 3-5 days.

Preferably, the preliminary identification method includes morphological feature identification and molecular identification.

Preferably, morphological characterization is performed based on characteristics of colonies, hyphae, sclerotium, conidia and conidiophores of the strain.

Preferably, when the storage time is short, the solid PDA slant culture medium inoculated with the sample to be tested is stored at 4 ℃.

Preferably, when the preservation time is long, the sample to be tested is preserved in 20% glycerol at-80 ℃.

Preferably, the genomic DNA extraction method comprises: performing activation culture on the preserved strain; obtaining mycelium to extract genome DNA; washing the DNA precipitate with ethanol and air-drying; the DNA was dissolved using 1xTE solution.

Preferably, the extraction method of the genomic DNA of the strain includes precipitation method, silica gel membrane mini-column method, silica gel chromatography column method, magnetic bead method.

Preferably, the activation culture conditions are PDA medium at 25℃for 7 days.

Preferably, the genomic DNA extraction method further comprises: grinding mycelium with liquid nitrogen, and adding the lysate to crack mycelium.

Preferably, the type of sample to be tested for extracting whole genomic DNA can be mycelium, sclerotium or spore fluid. Particularly preferably, the type of sample to be tested for extracting whole genomic DNA is mycelium.

Preferably, the precipitation of DNA in the method of extracting genomic DNA is washed with 60% to 80% ethanol. Particularly preferably, the precipitation of DNA in the method of extracting genomic DNA is washed with 70% ethanol.

Preferably, genomic DNA is extracted and then electrophoresed and the concentration of DNA is detected.

Preferably, the electric field strength of the electrophoresis is set to 8V/cm. Preferably, the electrophoresis time is 30 minutes.

Preferably, the dye of the DNA can be Ethidium Bromide (EB). Preferably, the dye of the DNA can be a 4S Green nucleic acid stain (4S Green nucleic acid stain).

Preferably, the step of constructing the genomic library comprises: cleavage of genomic DNA fragments, library construction, library quality control.

Preferably, the initial amount of genomic DNA used for pool building is 50-200 ng.

Preferably, the genomic DNA sample cleavage method includes restriction endonuclease cleavage, ultrasonic cleavage, chemical cleavage, and the like.

Preferably, the genomic DNA is cut into fragments of DNA ranging in size from 400 to 500 bp.

Preferably, the method of library construction comprises: plug-in library construction, linker library construction, and the like.

Preferably, an insertion library of fragments is constructed according to a library construction procedure. Specifically, library construction was performed according to the procedure provided by NEB Next Ultra II DNA Library Prep Kit.

Preferably, the prepared library is subjected to quality control before sequencing, and a qualified library is selected for sequencing. Preferably, the method of library quality control comprises: agarose gel electrophoresis, fluorescent quantitation, quality assessment, etc.

Preferably, the sequencing method is whole genome sequencing.

Preferably, whole genome sequencing employs high throughput sequencing techniques.

Preferably, the sequencing method uses Illumina HiSeq 2500 for whole genome sequencing of the library.

Preferably, the sequencing strategy selected for sequencing is PE-150.

Preferably, the sequence is aligned with the reference genome separately using GATK after quality control of the original sequence of the sample after sequencing. Preferably, the reference genome is b.cinerea B05.10.

Preferably, the comparison method is as follows: extracting target DNA sequence from the obtained whole genome DNA sequence of the sample to be detected, and comparing the target DNA sequence with the corresponding sequence in the reference genome. Preferably, the DNA sequence of interest is the RBP2 gene sequence.

Preferably, the sequence extraction method comprises the following steps: coordinates and gene sequences of the target DNA sequence fragments in the reference genome are obtained. Specifically, the DNA sequence fragment of interest is RPB2.

Preferably, the LSU has a coordinate in the reference genome of chromosome 4 nc_037313.1:3532-4394.

Preferably, the ITS has a coordinate in the reference genome of chromosome 4 NC_037313.1:4421-4957.

Preferably, the coordinates of RPB2 in the reference genome are chromosome 14 nc_037323.1:703646-704738.

Preferably, the alignment method is performed using Clustal W software.

Preferably, the alignment method uses FAFFT software for sequence alignment.

Preferably, the alignment method uses MUSCLE software for sequence alignment.

Preferably, the method employs a contiguity approach to construct a phylogenetic tree.

Specifically, phylogenetic trees were constructed using the molecular evolution genetic analysis software MEGA 6, in which the bootstrap analysis was performed using 1000 replicates.

Preferably, in the phylogenetic tree, the LSU sequence fragments, ITS sequence fragments and RPB2 genes of Botrytis species and exogroups are obtained from a common open database, wherein the common open database is NCBI (https:// www.ncbi.nlm.nih.gov /). Preferably, the sequence of the RPB2 gene of botrytis.

Preferably, the sample to be tested is a bacterial species collected from China rose.

The application provides application of the detection method in improving the accuracy of identifying the botrytis cinerea.

The application provides application of the detection method in the aspect of tracing the source of the Chinese rose botrytis.

The application provides an application of an RPB2 gene sequence obtained based on whole genome sequencing in the identification of Botrytis cinerea. Preferably, the Botrytis cinerea is one or more of Botrytis cinerea, botrytis petingonii and Botrytis fabae. Preferably, the Botrytis cinerea is Botrytis cinerea or Botrytis petingonii. Preferably, the Botrytis cinerea is Botrytis cinerea or Botrytis Fabae. Preferably, the Botrytis cinerea is Botrytis cinerea. Preferably, the Botrytis cinerea is Botrytis cinerea, botrytis petingonii, botrytis fabae.

Preferably, the RPB2 gene sequence obtained based on whole genome sequencing is shown in SEQ ID No.1 to SEQ ID No.3.

The beneficial effects are that:

in the prior art, in the identification method of plant pathogenic bacteria, the identification is usually carried out according to the form and physiological characteristics of the plant pathogenic bacteria, and then the identification is carried out by assisting with a biochemical test means, so that the operation of the identification process is complicated, the time is long, the obtained result is not clear in some cases, and the defect of inaccurate identification result exists. With the maturation of molecular biology technology, classification and identification methods based on high-throughput sequencing technology are widely applied to plant pathogenic fungi identification, and although the current research shows that the combination of RPB2 gene and other different gene fragments such as Internal Transcribed Spacer (ITS), heat shock protein 60 (HSP 60), glyceraldehyde-3-phosphate dehydrogenase (G3 PDH) and the like is applied to the classification and phylogenetic research of fungi, few species including B.cinerea cannot be effectively identified, and the result often lacks accuracy. The detection method provided by the application can be used for classifying and identifying the gray mold harmful pathogenic bacteria of China rose with higher accuracy.

The application identifies 100 bacterial strains obtained by collection and separation, obtains the full DNA sequence of the Chinese rose botrytis cinerea by a full genome sequencing technology, compares the full genome DNA, extracts the RPB2 gene sequence of a sample, constructs a phylogenetic tree by using the obtained SNP information, and obtains the RPB2 gene obtained based on the full genome sequencing to be highly specialized in B.cinerea according to the species identification result displayed by the phylogenetic tree. Specifically, the application utilizes LSU sequence fragments, ITS sequence fragments and RPB2 genes to classify Botrytis species. Phylogenetic trees based on ITS sequence fragments demonstrate that seven species including b.cinerea can be identified from botrytis using ITS sequence fragments, but that the species within this branch cannot be further identified. Although the ITS sequence fragment has a certain identification effect, the accuracy or precision of identification is insufficient, and more accurate results can be obtained by combining morphological characteristics or physiological characteristics and the like, which indicates that the ITS sequence fragment can be used for identifying a part of species in Botrytis. The ITS sequence fragments obtained based on whole genome sequencing cannot be further classified into seven categories in branches, so that the ITS sequence fragments obtained based on whole genome sequencing have a certain identification effect in classifying and identifying the inter-species relationship of Botrytis, but have insufficient accuracy, and if the morphological characteristics or the phenotypic characteristics are further combined to identify different species of Botrytis, the identification difficulty and the identification time are increased, so that the identification efficiency is lower.

The LSU sequence fragments obtained based on whole genome sequencing can distinguish the exoid group, but when the species relationship of Botrytis is identified in a classifying way, AN-02, B.cinerea and other Botrytis species in a phylogenetic tree have no definite branching mode, and the B.cinerea and other Botrytis species cannot be identified in a classifying way, namely, the LSU sequence fragments obtained based on whole genome sequencing are less effective in identifying the species relationship of Botrytis in a classifying way than ITS sequence fragments obtained based on whole genome sequencing. Thus, LSU sequence fragments cannot be used for identification between B.cinerea and various species within the genus.

The effectiveness and the accuracy of the RPB2 gene obtained based on whole genome sequencing in classifying and identifying the interspecies relationship of Botrytis are far higher than those of the ITS sequence fragment and the LSU sequence fragment. 100 B.cinerea samples are gathered together with three representative strains of LC14, AN-16 and AN-38 in a phylogenetic tree constructed based on RPB2 genes obtained by whole genome sequencing, which shows that all the 100 isolated strains separated and detected from China rose are B.cinerea and that the RPB2 genes obtained by whole genome sequencing can clearly distinguish several types of B.cinerea, B.fabae, B.eucalypti and B.pelargonii, and that the ITS fragments obtained by whole genome sequencing, LSU fragments obtained by whole genome sequencing and the combined analysis based on LSU sequence fragments, ITS sequence fragments and RPB2 genes cannot obtain the identification result. The above conclusion further illustrates that the RPB2 gene obtained based on whole genome sequencing is capable of efficiently and accurately identifying the species-to-species relationship of botrytis. The application discovers that the RPB2 gene is highly specialized to B.cinerea, so that the RPB2 single gene fragment obtained based on whole genome sequencing can provide enough information to determine branches so as to achieve the aim of accurate classification and identification. The RPB2 single gene fragment obtained based on whole genome sequencing is used as an advantageous tool for identifying other species in B.cinerea and Botrytis, and the identification accuracy is far higher than that of the detection method provided by the prior art.

Compared with the traditional methods of identifying ITS sequence fragments, LSU sequence fragments and RPB2 genes singly or jointly, the application obtains the result of highly specializing RPB2 genes of B.cinerea. The B.cinerea can be completely distinguished from other species of Botrytis by only using the RPB2 gene obtained by whole genome sequencing, and the obtained identification result is more accurate.

Further, in the prior art, the identification of the Botrytis cinerea is performed by using single sequence fragments or gene combination analysis, for example, the study of Zhang et al (2010) shows that the single use of ITS sequences cannot accurately identify the B.cinerea, B.sinoalii, B.porri, B.acroda, B.squarasa and B.byssoidea6 species, and the sequence similarity between the two species reaches 98.9-100%, so that the method cannot be used for identifying the B.cinerea; studies by Ferrada et al (2016) showed that neither ITS nor LSU sequence alone could accurately identify b.cinerea nor b.prunorum, both species having 100% sequence similarity, and this method could not be used to identify b.cinerea. In the past, because the identification effect of the single sequence fragment is poor, classification identification is carried out by improving a mode of gene joint analysis, for example, in research of separation identification (Zhang Shuangyan and the like) of gray mold pathogenic bacteria of China rose in Yunnan area, a phylogenetic tree is constructed by adopting ITS and RPB2 joint analysis, the identification result is that the similarity between 2 sequences of 87 strains and the sequence of B.cinerea is over 99 percent, the similarity between 2 sequences of 1 strains and the sequence of B.fabae is over 99 percent, and pathogenic bacteria cannot be completely identified. Specifically, increasing the number of sequence fragments for identifying pathogenic bacteria can increase the accuracy of the identification result, but the prior art uses, for example, ITS, HSP60, and G3PDH; the ITS and RPB2 analysis also cannot be completely distinguished. Furthermore, the ITS sequence fragment and the LUS sequence fragment obtained by whole genome sequencing in example 2 of the present application also cannot completely distinguish B.cinerea from other species of Botrytis. Whereas the RPB2 gene obtained by whole genome sequencing is able to completely distinguish b.cinerea from other species of botrytis. Compared with the direction of adopting combined gene analysis in the prior art, the method reduces the number of sequence fragments for identifying pathogenic bacteria, but increases the accuracy of identification results, which indicates that not all sequence fragments for identifying pathogenic bacteria obtained by using whole genome sequencing are suitable for identifying B.cinerea, and the RPB2 gene obtained by using whole genome sequencing is particularly suitable for identifying B.cinerea.

Therefore, the application utilizes the RPB2 gene obtained by whole genome sequencing to identify the B.cinerea, thereby greatly improving the accuracy of the RPB2 gene sequence and improving the classification identification efficiency. The discovery of the application reveals that the pathogenic bacteria causing the Chinese rose gray mold disease in the Yunnan area is B.cinerea for the first time, and the whole genome sequencing is adopted to obtain the RPB2 gene sequence, so that the B.cinerea can be rapidly and accurately identified, and the detection method provided by the application can provide reference for the control and pathogen identification of the Chinese rose gray mold in the Yunnan area.

Drawings

FIG. 1 is a phylogenetic tree based on ITS fragments provided by the present application;

FIG. 2 is a phylogenetic tree based on LSU fragments provided by the present application;

FIG. 3 is a phylogenetic tree based on the RPB2 gene provided by the application.

Detailed Description

The following detailed description refers to the accompanying drawings.

With the enrichment and perfection of the DNA sequence fragment data of Botrytis species, the application discovers that the RPB2 gene obtained by utilizing whole genome sequencing can effectively identify B.cinerea. The application identifies 100 bacterial strains obtained by collection and separation, and the results show that the bacterial strains are B.cinerea. The discovery reveals that the pathogenic bacteria causing the ash mildew disease of China rose in the Yunnan area is B.cinerea for the first time, so that the B.cinerea can be accurately identified by utilizing the RPB2 gene sequence obtained by whole genome sequencing. The method not only simplifies the experimental flow and improves the identification efficiency, but also greatly reduces the identification cost, and can provide reference for the control and pathogen identification of the China rose gray mold in the Yunnan area.

It should be noted that AN-02, AN-16 and AN-38 represent strains isolated from the 2 nd, 6 th and 38 th sampling points of the Anning region of Kunming city, respectively, and LC14 represents strain isolated from the 14 th sampling point of the Malz region Chu Zhou. The sequence of AN-16 corresponds to SEQ ID No.1. The sequence of AN-38 corresponds to SEQ ID No.2. The sequence of LC14 corresponds to SEQ ID No.3. Sequence number KX867998 of B.cinerea in NCBI corresponds to SEQ ID No.4.Botrytis cinerea is also denoted b.cinerea in the present application.

Example 1

The specific method for the test is as follows:

1. bacterial strain origin

Long-term and widespread planting of rose cut flowers in the Yunnan region provides favorable conditions for colonization by b.cinerea. In the embodiment, cut flower China rose tissues infected with the gray mold disease collected from main production areas of different China rose cut flowers in Yunnan are brought back to a laboratory, pathogen is separated after the cut flower China rose tissues are cultured for 3 to 5 days in dark conditions with the temperature of 25 ℃ and the relative humidity of 80%, and pure culture separated strains are obtained through multiple purification and single spore separation.

Under laboratory culture conditions, B.cinerea was initially identified based on morphological features of colonies, hyphae, sclerotium, conidia and conidiophores of the strain. During 2018-2020, a total of 609 field b.cinerea strains were obtained and 100 were selected from different production areas and hosts for molecular characterization. The strain was stored for a short period of time on solid PDA slant medium at 4℃and for a long period of time in 20% glycerol at-80 ℃.

2. Genomic DNA extraction and detection

Taking out the 100 strains, performing activation culture on PDA culture medium at 25deg.C, scraping mycelia into 2ml centrifuge tube after 7 days.

During extraction, mycelium is taken out and put into a mortar, a small amount of liquid nitrogen is added for rapid and full grinding, and a lysis buffer is added according toThe method described by et al (1992) extracts DNA. The DNA precipitate was washed with 70% ethanol and air dried, and finally dissolved with 100. Mu.l of 1xTE solution. The dissolved DNA was electrophoresed in TAE buffer with 0.8% (w/V) agarose gel, and after 30 minutes (8V/cm), the DNA was stained with 4S green nucleic acid stain (Sangon Biotech Co., ltd.) and examined by photographing. DNA concentrations were measured using a Nanodrop ND-2000 spectrophotometer (Thermo FisherScientific Inc., waltham, mass., USA). Finally, the genomic DNA was stored at-80℃and used for the next library experiments.

3. Library construction and sequencing

The initial amount of genomic DNA used for pool building in this example was 50 to 200ng and the final volume was 50. Mu.l. Genomic DNA samples were cut into fragments of 400-500 bp DNA in covarias microtubes using covarias M220 sonicator (covarias llc., MA, USA). An insertion library of these fragments was constructed according to the protocol of NEB Next Ultra IIDNA Library Prep Kit (New England Biolabs inc., MA, USA).

The prepared library was quality controlled using an Agilent 2100 Bioanalyzer analyzer (Agilent Technologies inc., CA, USA) prior to sequencing. The qualified library was sequenced on an Illumina HiSeq 2500 platform (Illumina inc., CA, USA) using the PE-150 strategy.

SNP detection and sequence extraction

And (3) obtaining the original sequence data of 100 B.cinerea samples through second generation sequencing, and respectively comparing the sequences with a reference genome B.cinerea B05.10 by adopting GATK after quality control of the original sequences. Coordinates of the LSU sequence fragment, ITS sequence fragment and RPB2 gene in the reference genome are chromosome 4 nc_037313.1:3532-4394, chromosome 4 nc_037313.1:4421-4957 and chromosome 14 NC_037323.1:703646-704738.

NCBI homologous sequence download and phylogenetic Tree construction

The LSU sequence fragments, ITS sequence fragments and RPB2 genes of other Botrytis species and exogroups were downloaded from the public open database NCBI (https:// www.ncbi.nlm.nih.gov /). The 3 DNA sequence fragments were aligned separately using the Clustal W program and phylogenetic trees were constructed using the adjacency method in the molecular evolution genetic analysis software MEGA 6 (Tamura, 2011), in which the bootstrap analysis was performed using 1000 replicates.

Example 2

This example shows the analysis result of example 1.

The phylogenetic tree based on the ITS sequence fragment is shown in figure 1, and the support rate of bootstrap in the phylogenetic tree is over 95 percent, which indicates that the phylogenetic tree has high reliability. Preferably, the ITS sequence is obtained by high throughput sequencing techniques. As shown in the A, B correspondence of FIG. 1, 100 samples are collected on two end branches, 25 samples and 75 samples, respectively, represented by AN-02 and AN-16, respectively, where AN-02 and B.cinerea MT968495 are collectively collected on branch A, AN-16, B.cinerea OU989292 and B.cinerea MK558826, and six other species (B.pseudobulb, B.sinovicola, B.pelargoni, B.fabae, B.eucalypti and B.carolina) are collected on branch B. Specifically, AN-16, B.pseudosporieea MF461632, B.sinosporila JN692381, B.pseudosporieea JN692379, B.pelargonii MT477708, B.pelargonii KX987153, B.fabae MK217911, B.fabae EU563125, B.eucalypti MF996367, B.eucalypti KX301017, B.cinerea OU989292, B.cinerea MK558826, B.caroliniana MN105496, B.caroliniana MG886399 in branch B could not be identified. The adjacent A and B branches are gathered together into a larger branch. As shown in fig. 1, the larger branch, which A, B clusters, is well differentiated from the other branches, and the A, B branch can distinguish between the outer group and seven species including b.cinerea from botrytis, but the species within the branch cannot be identified. The results show that the ITS sequence fragments have a certain identification effect, and in addition, the ITS sequence fragments can be used for further identification in combination with morphological and phenotypic characteristics and the like, for example, B.fabae and B.cinerea are gathered together on the phylogenetic tree of the ITS, but B.fabae is an obligatory parasitic fungus, and the host range is limited to a few species of leguminous plants and is obviously different from B.cinerea with a wide host range. Pelargonii was reported in pelargonium kunminum, indicating that b.pelargonii is a pelargonium obligatory parasitic fungus and b.cinerea can be identified by ITS identification in combination with morphological features. Carolina is pathogenic to blackberry fruits and broad bean leaves, indicating that b.carolina is an obligatory parasitic fungus and b.cinerea can be identified by ITS identification combined with morphological features.

The phylogenetic tree based on LSU sequence fragments is shown in FIG. 2, with all 100 samples grouped together as AN-02. Preferably, the LSU sequence is obtained by high throughput sequencing techniques. In the evolutionary tree shown in FIG. 2, B.cinerea can be distinguished from the outer group, but AN-02, B.cinerea and other Botrytis species are randomly dispersed in the phylogenetic tree, and cannot be classified and identified without a clear branching pattern. The results of FIG. 2 show that AN-02, B.galand MH867353, B.hyachinthi MH867354, B.cinerea MW474943, B.acroda MH866244, B.cinerea MN148533, B.cinerea MT781367, B.cinerea ON090417, B.globosa MH869245, B.acroda JN938890, B.cinerea KP671724, B.narciscolia MH867815 are not further distinguishable. Except for the group of species (m.fructigenaay 544683, m.fructigena MH868240, s.minor MH866179, S bulboummh 866668) and b.criptiome MH868294, the relatedness between species could not be inferred from this fragment, as shown in fig. 2, so LSU sequence fragments could not identify b.cinerea and between the species.

Phylogenetic tree constructed based on RPB2 gene obtained by whole genome sequencing contains 100 b.cinerea samples, 48 botrytis samples (including the 36 classification units widely recognized at present) and 2 outer clusters (Monilinia fructigena and Sclerotinia sclerotiorum). Preferably, whole genome sequencing is a high throughput sequencing technique. In fig. 3, monilinia fructigena is denoted as m.fructigena AJ745715, sclerotinia sclerotiorum is denoted as s.sclerotiorum AJ745716. The phylogenetic tree results shown in FIG. 3 show that 100 samples were pooled on three terminal branches, 9, 7 and 84 individuals, respectively, represented by LC14, AN-16 and AN-38, respectively, and three representative strains were pooled with B.cinerea, as shown in FIG. 3, indicating that all 100 isolates isolated from China rose were B.cinerea. Specifically, the accuracy of the RPB2 gene obtained by whole genome sequencing reaches 100% when B.cinerea is identified, which is far higher than the accuracy of ITS sequence fragments and LSU sequence fragments in the aspect of B.cinerea identification.

Specifically, the RPB2 gene obtained based on whole genome sequencing can distinguish B.cinerea, B.pseudosporea, B.fabae, B.eucalypti and B.pelargoni; in the classification and identification of B.cinerea, LC14, B.cinerea JN692417 and B.cinerea OP623527 are gathered together to form a branch together with AN-16, and branches of strains represented by LC14 and AN-16 are finally gathered together with AN38, B.cinerea AJ745677 and B.cinerea AJ745675, which indicate that the single gene fragment of RPB2 obtained based on whole genome sequencing can be used for effectively identifying B.cinerea.

Example 3

The past research results show that the method for constructing phylogenetic tree by adopting the combination or the independent of three gene sequences of RPB2, G3PDH and HSP60 can identify most species of Botrytis, but the accuracy of identification is far lower than that of the detection method provided by the application. The results obtained by sequencing identification after PCR amplification in the prior art are not ideal, for example, the study of Zhang et al (2010) shows that the ITS sequence alone cannot accurately identify 6 species of B.cinerea, B.sinoallii, B.porri, B.acroda, B.squarasa and B.byssoide, the sequence similarity between the species reaches 98.9-100%, and the method cannot be used for identifying B.cinerea; studies by Ferrada et al (2016) showed that neither ITS nor LSU sequence alone could accurately identify b.cinerea and b.prunorum, both species were 100% similar in sequence and indistinguishable. According to the study of Staats et al (2005), the following is used aloneThe RPB2 sequence cannot accurately identify B.cinerea, B.pelargonii and B.fabae; neither G3PDH nor HSP60 sequences alone can accurately identify B.cinerea and B.pelargonii. And the combination analysis of three gene sequences of RPB2, G3PDH and HSP60 can not accurately identify the B.cinerea, B.eucalypti, B.pelargonii and B.fabae4 species (Brauna-Etc., 2023; garfinkel,2021; kevin et al, 2014). These results further demonstrate the high accuracy and effectiveness of the RPB2 gene obtained based on whole genome sequencing provided by the present application in classifying and identifying the intervarietal relationship of botrytis.

Furthermore, from the results provided in example 2, it was found that the ITS fragments obtained based on whole genome sequencing are able to distinguish the exoid group, which is expressed in the classification of the species relationship of Botrytis: seven species including B.cinerea can be identified from Botrytis, but the seven species in the branches cannot be further classified, so that ITS sequence fragments obtained based on whole genome sequencing have a certain identification effect in classifying and identifying the species-to-species relationship of Botrytis, but the accuracy of the identification result is insufficient.

The LSU sequence fragments obtained by whole genome sequencing can distinguish the outer group. However, when the species relationship of Botrytis is classified and identified, the AN-02, B.cinerea and other Botrytis species in the phylogenetic tree have no definite branching mode, and the B.cinerea and other Botrytis species cannot be classified and identified. LSU sequence fragments obtained based on whole genome sequencing are less effective in classifying and identifying the intervarietal relationships of Botrytis than ITS sequence fragments obtained based on whole genome sequencing.

The effectiveness and the accuracy of the RPB2 gene obtained by whole genome sequencing in the classification and identification of the species-to-species relationship of Botrytis are far higher than those of the ITS sequence fragment and the LSU sequence fragment in the classification and identification of the species-to-species relationship of Botrytis. 100 B.cinerea samples are gathered together with three representative strains of LC14, AN-16 and AN-38 in a phylogenetic tree constructed based on RPB2 genes obtained by whole genome sequencing, which shows that all the 100 separated strains separated and detected from China rose are B.cinerea, and the RPB2 genes obtained by whole genome sequencing can clearly distinguish several types of B.cinerea, B.fabae, B.eucalypti and B.pelargonii, while the combination analysis of ITS sequence fragments obtained by whole genome sequencing, LSU sequence fragments obtained by whole genome sequencing and the combination analysis of LSU sequence fragments, ITS sequence fragments and RPB2 gene sequences cannot obtain the identification result, and the results further show that the RPB2 genes obtained by whole genome sequencing can effectively and accurately identify the species relationship of Botrytis. The application discovers that the RPB2 gene is highly specialized to B.cinerea, so that the RPB2 single gene fragment obtained based on whole genome sequencing can provide enough information to determine branches so as to achieve the aim of accurately classifying and identifying, and the accuracy of identification is far higher than that of detection methods provided by the prior art by taking the RPB2 single gene fragment obtained based on whole genome sequencing as a favorable tool for identifying other species of B.cinerea and Botrytis.

The embodiment provides application of a detection method of the botrytis cinerea in the aspect of tracing the botrytis cinerea. Phylogenetic tree analysis results show that phylogenetic tree constructed based on RPB2 gene obtained by whole genome sequencing shows that 100 B.cinerea samples are clustered with three representative strains of LC14, AN-16 and AN-38. Further, LC14, B.cinerea JN692417 and B.cinerea OP623527 are gathered together to form a branch, and then gathered together with AN-16, and branches of strains represented by LC14 and AN-16 are gathered together with AN-38, B.cinerea AJ745677 and B.cinerea AJ745675, wherein the self-development supporting rate is 99%, so that the separated pathogenic bacteria can be determined to be B.cinerea. According to a preferred embodiment, specific SNP sites are obtained by comparing the whole genome DNA sequence of a sample to be tested obtained by whole genome sequencing with the standard sequence of the RPB2 gene of Botrytis cinerea. And analyzing specific SNP loci in the RPB2 gene obtained by whole genome sequencing to obtain the traceability information of the gray mold of China rose. Preferably, the traceability information of the trichosanthes kirilowii can be obtained based on SNP-based population genetic analysis and/or SNP-based phylogenetic tree analysis. In particular, the traceability information can include the origin and host information of the pathogenic bacteria. According to the traceability information, the evolution rule of pathogenic bacteria, the differentiation condition of pathogenic bacteria, the drug resistance condition and the like can be researched, so that guiding significance is provided for preventing and controlling the gray mold of China rose in the Yunnan area.

In this example, the botrytis cinerea is referred to as botrytis cinerea, and the botrytis cinerea is referred to as China rose botrytis cinerea.

It should be noted that the above-described embodiments are exemplary, and that a person skilled in the art, in light of the present disclosure, may devise various solutions that fall within the scope of the present disclosure and fall within the scope of the present disclosure. It should be understood by those skilled in the art that the present description and drawings are illustrative and not limiting to the claims. The scope of the application is defined by the claims and their equivalents. The description of the application includes a plurality of inventive concepts, such as "preferably", "according to a preferred embodiment" or "optionally" each meaning that the corresponding paragraph discloses a separate concept, the applicant reserves the right to filed a divisional application according to each inventive concept. Throughout this document, the word "preferably" is used in a generic sense to mean only one alternative, and not to be construed as necessarily required, so that the applicant reserves the right to forego or delete the relevant preferred feature at any time.

Claims

1. The detection method of the China rose botrytis is characterized by comprising the following steps:

obtaining the nucleotide sequence of the whole genome DNA of the sample to be detected by using a whole genome sequencing technology;

performing RPB2 gene comparison on the sample to be tested;

and judging whether the sample to be tested contains the Chinese rose gray mold bacteria or not according to the comparison result.

2. The method of claim 1, wherein the method of comparing is: and comparing the RPB2 gene sequence extracted by the whole genome sequencing of the sample to be detected with the standard sequence of the RPB2 gene of the Botrytis cinerea.

3. The method according to claim 1 or 2, wherein when the sample to be tested has an RPB2 gene and the RPB2 gene contains a specific SNP site, the sample to be tested is indicated to belong to Botrytis cinerea, wherein,

the nucleotide sequence of the RPB2 gene of the sample to be detected is shown in SEQ ID No.1 to SEQ ID No.3.

4. The method according to any one of claims 1 to 3, wherein the SNP site comprises:

5. The method of any one of claims 1 to 4, wherein the whole genome sequencing technique is a high throughput sequencing technique.

6. The method according to any one of claims 1 to 5, wherein the type of the sample to be tested for extracting whole genome DNA is mycelium, sclerotium or spore fluid.

7. The use of the detection method according to any one of claims 1 to 6 for improving the accuracy of identification of botrytis cinerea.

8. The use of the detection method according to any one of claims 1 to 7 in the traceability of the botrytis cinerea.

9. The application of the RPB2 gene sequence obtained based on whole genome sequencing in the identification of the Botrytis cinerea.

10. The use according to claim 9, wherein the RPB2 gene sequence obtained based on whole genome sequencing is shown in SEQ ID No.1 to SEQ ID No.3.